Abstract Integrating different sensory inputs, including visual, auditory and somatosensory stimuli, is required for rapid environmental perception and response. Failure to correctly develop Multisensory Integrating Neurons (MINs) would lead to difficulties in properly orienting towards and/or responding to relevant environmental stimuli. Therefore, it is important to study how MINs develop and function at the molecular level. Multisensory stimuli are integrated in the superior colliculus (SC) of the mammalian midbrain and in the optic tectum (OT) of non-mammalian species. Electrophysiological recordings of SC neurons in cats and monkeys reveal that sensory integration in the SC occurs in MINs of the deep layers of the SC, which amplify or dampen responses from cross-modal sensory inputs depending on their temporal and spatial proximity. Although the presence of MINs is supported by electrophysiological recordings, nothing is known about the molecular mechanisms that oversee MINs development, specification and neurocircuitry formation that ultimately lead to their unique function. Our study is designed to fill this gap in the current understanding by identifying and characterizing, in a genetically tractable model organism such as zebrafish, MINs that integrate visual and auditory input. This work will establish the necessary groundwork to begin to study the molecular mechanisms overseeing MINs development and function. The zebrafish OT is made of a cellular layer consisting of neuronal cell bodies and a neuropil layer consisting of neuronal projections. Two types of neurons are predominately found in the tectum: periventricular interneurons (PVIN), which are unipolar neurons that synapse at different layers within the neuropil, and periventricular projection neurons (PVPN) that have dendrites in the neuropil layer and an axon extending out of the tectum into motor areas such as the reticulospinal formation. We have adapted different cutting-edge technologies, including optogenetic and cell labeling techniques to detect by calcium imaging the presence of auditory-visual (AV) MINs in the zebrafish OT and characterize their morphology using photoconvertible GFP. Characterization of one such AV MIN shows that it is a PVIN with projections extending in neuropil areas consistent with receiving visual and auditory inputs. In Aim 1 of this proposal, we would like to determine where in the zebrafish OT, AV MINs are localized, what is their morphology and what type of interneurons they are as determined by the neurotransmitters they release. In Aim 2, we would like to test if the AV MINs are capable of integrating visual and auditory inputs and not merely respond to the individual stimuli. Finally, in Aim 3, we seek to understand the molecular signatures of AV MINs by obtaining their transcriptional profiles.